Enhanced secretion of a polypeptide by a microorganism

ABSTRACT

Described herein are methods for the enhanced production of secreted proteins. The secretion of a protein of interest having a substantially non-polar carboxy tail is enhanced by the placement of charged amino acid residues at the carboxy terminus either by adding to the native peptide or by replacing, i.e., substituting, the terminal residues of the native peptide.

This is a Divisional of U.S. patent application Ser. No. 09/975,132,filed on Oct. 9, 2001 now U.S. Pat. No. 6,846,653, which claims priorityto U.S. Provisional Patent Application Ser. No. 60/239,531, filed Oct.10, 2000.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), the present application claims benefit ofand priority to U.S. Ser. No. 60/239,531, entitled “Enhanced Secretionof a Polypeptide by a Microorganism”, filed Oct. 10, 2000, by MarcKolkman.

FIELD OF THE INVENTION

This invention relates to the production and secretion of a selectedpolypetide. More particularly, the present invention provides for theenhanced secretion of a selected polypeptide by a microorganism, such asa Bacillus species.

BACKGROUND OF THE INVENTION

Eubacteria export numerous proteins across the plasma membrane intoeither the periplasmic space (Gram-negative species), or the growthmedium (Gram-positive species). The Gram-positive eubacterium Bacillussubtilis and, in particular, its close relatives Bacillusamyloliquefaciens and Bacillus licheniformis are well known for theirhigh capacity to secrete proteins (at gram per liter concentrations)into the medium. This property, which allows the efficient separation of(secreted) proteins from the bulk cytoplasmic protein complement, hasled to the commercial exploitation of the latter bacilli as important“cell factories.” Despite their high capacity to secrete proteins ofGram-positive origin, the secretion of recombinant proteins fromGram-negative eubacterial or eukaryotic origin by Bacillus species isoften inefficient.

General strategies for the secretion of heterologous proteins by bacilliare based on the in-frame fusion of the respective protein with anamino-terminal signal peptide that directs this protein into a secretionpathway, for example the Sec-dependent secretory pathway. Upontranslocation across the membrane, the signal peptide is removed by asignal peptidase, which is a prerequisite for the release of thetranslocated protein from the membrane, and its secretion into themedium.

Proteolysis in bacteria serves to rid the cell of abnormal, andmisfolded proteins. A unique mechanism for the destruction of abnormalproteins resulting from abortive termination of translation is providedby the SsrA-mediated tagging and degradation system (for a recentreview, see Karzai et al. (2000). The SsrA-SmpB system for proteintagging, directed degradation and ribosome rescue. Nat. Struct. Biol.7:449-455.). SsrA, also called 10Sa RNA or tmRNA is a highly conservedRNA molecule in eubacteria. It is a unique molecule that can act as botha tRNA and an mRNA in a process referred to as trans-translation (Atkinset al. 1996. A case for trans translation. Nature 379:769-771, Jentsch1996. When proteins receive deadly messages at birth. Science271:955-956, Keiler et al. 1996. Role of a peptide tagging system indegradation of proteins synthesized from damaged messenger RNA. Science271:990-993). This mechanism provides the cell a way to releaseribosomes that are stalled on untranslatable mRNAs, e.g. mRNAs lackingin-frame stop codons. In the model for SsrA action, SsrA charged withalanine enters the A site of a stalled ribosome, mimicking a tRNA. Thealanine is added to the uncompleted polypeptide chain; and then, servingas an mRNA, SsrA provides a short reading frame followed by a stop codonas a template to add a short peptide to the nascent polypeptide beforetranslation terminates and a tagged protein is released. The peptide tag(encoded by SsrA) functions as a proteolytic degradation signal, and inEscherichia coli four proteases have been identified that degradeproteins tagged by SsrA. ClpXP, ClpAP, and FtsH (HflB) degrade SsrAtagged proteins in the cytoplasm (Gottesman et al. 1998. The ClpXP andClpAP proteases degrade proteins with carboxy-terminal peptide tailsadded by the SsrA-tagging system. Genes Dev. 12:1338-1347, Herman, etal. 1998. Degradation of carboxy-terminal-tagged cytoplasmic proteins bythe Escherichia coli protease HflB (FtsH). Genes Dev. 12:1348-1355),while SsrA tagged proteins with signal peptides that are exported to theperiplasm of E. coli are degraded by Tsp (Prc) protease (Keiler et al.1996).

Protein production and secretion from Bacillus species is a majorproduction tool with a market of over $1 billion per year. However,proteolysis of proteins by endogenous proteases diminishes theproduction capability of these microorganisms. Thus, it would bebeneficial to have an mechanism for the enhanced production andsecretion proteins. The present invention provides such an advantage bychanging the nonpolar C-terminus of a protein by adding charged, polarresidues (or by replacing amino acids), so that the proteins areprotected against the bacillus proteases that degrade SsrA-taggedproteins.

SUMMARY OF THE INVENTION

Provided herein are methods for the enhanced production of peptides in ahost cell.

In one aspect of the invention, the present invention provides methodsfor increasing secretion of proteins from host microorganisms. In oneembodiment of the present invention, the protein is homologous ornaturally occurring in the host microorganism. In another embodiment ofthe present invention, the protein is heterologous to the hostmicroorganism. Accordingly, the present invention provides a method forincreasing secretion of a protein in a host cell using an expressionvector comprising nucleic acid sequence encoding a protein of interestwherein said nucleic acid sequence is under the control of expressionsignals capable of expressing said protein of interest in a hostmicroorganism; introducing the expression vector into a hostmicroorganism capable of expressing said protein and culturing saidmicroorganism under conditions suitable for expression of said secretionfactor and secretion of said protein.

In one embodiment, the host cell is transformed with a first DNAsequence encoding a signal peptide operably linked to a second DNAsequence encoding a protein. Said protein may be, but not limited to,hormones, enzymes, growth factors, cytokines, antibodies and the like.In another embodiment, the enzyme includes, but is not limited tohydrolases, such as protease, esterase, lipase, phenol oxidase,permease, amylase, pullulanase, cellulase, glucose isomerase, laccaseand protein disulfide isomerase. The second DNA sequence may encode aprotein that has been modified such that its carboxy-terminus possessesat least one, preferably two, charged amino acids. Such modification maybe by substitution of the native carboxy-terminal residues or additionof a tag sequence to the native protein's carboxy-terminus.

Further provided herein is a method of enhancing resistance toproteolysis of a protein. In a preferred embodiment the protein is asecreted protein. It is contemplated that the protein will comprise atag wherein the tag comprises at least one charged amino acid residue.The charged amino acid residue may be either a positively chargedresidue or it may be a negatively charged residue.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A. Northern blot of total RNA of B. subtilis 168 and B. subtilis168 ΔssrA, hybridized with an ssrA specific probe. At the bottom: thelevel of 16S RNA in both RNA samples. B. Growth curves of B. subtilis168 (---∘---) and B. subtilis 168 ΔssrA (-●-) at 37° C. in TSB medium.C. Growth of B. subtilis 168 and B. subtilis 168 ΔssrA on HI-agar platesat 25° C. or 45° C.

FIG. 2. hIL-3 expressed from an mRNA without a stop codon (pLATIL3TERM),accumulates in the medium of B. subtilis lacking SsrA (lanes 3, 6, 9),but not in cells containing functional SsrA (lanes 2, 5, 8). At threedifferent growth stages, samples were collected from cultures of B.subtilis 168 (pLATIL3) [lanes 1, 4, 7], B. subtilis 168 (pLATIL3TERM)[lanes 2, 5, 8], and B. subtilis 168 ΔssrA (pLATIL3TERM) [lane 3, 6, 9].After centrifugation, the proteins in the culture supernatants wereconcentrated by TCA precipitation and analyzed by SDS-PAGE and Westernblotting with anti-hIL-3 antibodies. The amount of total extracellularprotein of B. subtilis 168 (pLATIL3) that was applied to the gel [lanes1, 4, 7] was 10 times less then that of B. subtilis 168 (pLATIL3TERM)[lanes 2, 5, 8] or B. subtilis 168 ΔssrA (pLATIL3TERM) [lanes 3, 6, 9].M indicates a lane with a prestained protein ladder; the molecularweight of the upper band corresponds to 20 kDa, that of the lower bandto 15 kDa.

FIG. 3. Stability of hIL-3 variants with different C-terminal tags.

(A). Western blot analysis of hIL-3 protein variants produced by B.subtilis 168 transformed with plasmid pLATIL3 (lane 1), pLATIL3BStag(expression of hIL-3 with a C-terminal B. subtilis SsrA tag (AA-tag):hIL-3-AGKTNSFNQNVALAA (SEQ ID NO:1); lane 2), pLATIL3DDtag (expressionof hIL-3 with a DD-tag: hIL-3-AGKTNSFNQNVALDD (SEQ ID NO:2); lane 3),and pLATIL3ECtag (expression of hIL-3 with a C-terminal E. coil SsrA tag(EC-tag): hIL-3-AANDENYALAA (SEQ ID NO:3); lane 4). Culture supernatantsof cells entering the stationary phase were collected and analyzed bySDS-PAGE and Western blotting with anti-hIL-3 antibody.

(B). Pulse-chase assays: Cells of B. subtilis 168 (pLATIL3BStag) and 168(pLATIL3DDtag) were labeled with [³⁵S]-methionine for 1′ prior to chasewith excess non-radioactive methionine. Samples were withdrawn at thetimes indicated, centrifuged and the culture supernatants were analyzedby SDS-PAGE and fluorography.

(C). The amounts of hIL-3-AAtag and hIL3-DDtag in (B) were quantified bydetermination of the radioactivity in the dried gel using aPhosphorImager (Molecular Dynamics) and plotted.

FIG. 4. The ‘major extracellular proteases’ of B. subtilis play a rolein, the degradation of extracellular, SsrA-tagged h-IL3. Western blotanalysis of hIL-3 protein secreted by B. subtilis 168 harboring plasmidpLATIL3 (lane 1, 6) or pLATIL3TERM (lane 2, 7), and B. subtilis WB600 (amultiple protease negative strain) containing plasmid pLATIL3TERM andexpressing either wild-type SsrA (lane 3, 8), no SsrA (lane 4, 9) orSsrA^(DD) (lane 5, 10). Culture supernatants of cells entering thestationary phase were collected, concentrated by TCA precipitation,analyzed by SDS-PAGE and immunoblotting with anti-hIL-3 antibody. (lanes1-5) or anti-Bs-SsrAtag antibody (lanes 6-10). SsrA-tagged hIL-3 (lanes3, 5, 8, 10), run-off hIL-3 translation product (lane 4, and possiblyalso in lane 3 and 5, see text), and wild-type hIL-3 (lane 1) areindicated by the arrows (→). Protein bands with lower molecular weightthat also react with anti-hIL-3 antibody are supposedly degradationproducts of hIL-3, SsrA-tagged hIL-3 or run-off hIL-3 translationproduct.

FIG. 5. B. subtilis CtpA has an additional role in the degradation ofSsrA-tagged hIL-3. Western blot analysis of hIL-3 protein secreted by B.subtilis WB600 harboring plasmid (pLATIL3TERM) and carrying either noadditional mutation (lane 1, 6), or lacking CtpA (lane 2, 7), YvjB (lane3, 8), ClpP (lane 4, 9), or SsrA (lane 5, 10). Culture supernatants ofcells entering the stationary phase were collected, concentrated by TCAprecipitation, analyzed by SDS-PAGE and Western blotting with anti-hIL-3antibody (lane 1-5) or anti-Bs-SsrAtag antibody (lane 6-10). Thestraight arrows (→) mark SsrA-tagged hIL-3 (lanes 1-4 and lanes 6-9),and run-off translation product (lane 5 and possibly (see text) also inlanes 1-4). Degradation products of (SsrA-tagged) hIL-3 are indicated by˜>.

FIG. 6. Tagging of native B. subtilis proteins. Total intracellular orextracellular proteins produced by cells in the exponential growth phaseor stationary phase of B. subtilis 168 expressing wild-type SsrA(AA),168 IssrA^(DD) expressing SsrA^(DD) (DD), or 168 ΔssrA containing noSsrA RNA (−) were analyzed by Western blotting using anti-Bs-SsrAtagantibody.

FIG. 7. A native protein and examples of the types of tags encompassedby the instant invention. (A) Depicts the sequence of humaninterleukin-3 (SEQ ID NO:25); the (native) signal peptide is in bold.(B) Depicts the IL-3 sequence encoded by plasmid pLATIL3 (SEQ ID NO:26).The sequence of AmyL[ss]-interleukin-3 is for hIL-3 secretion inBacillus: the AmyL signal peptide is in bold, and this expressed IL-3lacks the last four amino acids (LAIF) of native hIL-3. The tag is initalics. (C) Depicts IL-3 with a tag that is a substitution of thenative protein's terminal two amino acids (SEQ ID NO:27). The tag is initalics. (D) Depicts a tag that is an addition to the native protein'scarboxy terminus (SEQ ID NO:28). Here the sequence of hIL3 as encoded bypLATIL3 with the SsrA-DD tag at the C-terminus [hIL3-DD] (signal peptidein bold, C-terminal tag in italics) is shown.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

The present invention provides for a process to enhance the productionof a desired secreted polypeptide by a suitable host cell. Inparticular, the present invention may be applicable to increase proteinproduction by B. subtilis. Changing the last two C-terminal amino acidsresidues into at least one, preferably two, charged amino acid residuesor adding at least one, preferably two, charged amino acid residues tothe COOH-terminus of a protein may be used to increase the yield of anyprotein secreted by B. subtilis. A longer tag sequence may be utilizedin the present invention. Especially the secretion of proteins that havea pI value >7 may be improved by this concept. In general, the minoralteration (adding or replacing two amino acid residues) itself shouldnot lead to a dramatic change in e.g. the specific activity of an enzymeor the thermostablity.

Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. The headings provided herein are notlimitations of the various aspects or embodiments of the invention whichcan be had by reference to the specification-as a whole. Accordingly,the terms defined immediately below are more fully defined by referenceto the specification as a whole.

Host Cell

“Host cell” means a cell that has the capacity to act as a host andexpression vehicle for an expression cassette according to theinvention. In one embodiment, the host cell is a microorganism. In apreferred embodiment according to the present invention, “host cell”means the cells of Bacillus. As used herein, the genus Bacillus includesall members known to those of skill in the art, including but notlimited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. ciculans, B. lautus and B. thuringiensis. Other cells useful in thepresent invention include Acinetobacter, Thermus, DeinococcusRadiodurans.

Polypeptide or Protein

The term “polypeptide” as used herein refers to a compound made up ofamino acid residues linked by peptide bonds. The term “protein” as usedherein may be synonymous with the term “polypeptide” or may refer, inaddition, to a complex of two or more polypeptides.

Additionally, a “protein of interest” or “polypeptide of interest”refers to the protein to be expressed and secreted by the host cell. Theprotein of interest may be any protein that up until now has beenconsidered for expression in prokaryotes. The protein of interest may beeither homologous or heterologous to the host.

The term “chimeric polypeptide” and “fusion polypeptide” are usedinterchangeably herein and refer to a protein that comprises at leasttwo separate and distinct regions that may or may not originate from thesame protein. For example, a signal peptide linked to the protein ofinterest wherein the signal peptide is not normally associated with theprotein of interest would be termed a chimeric polypeptide or chimericprotein.

Signal Sequence

A “signal peptide” as used herein refers to an amino-terminal extensionon a protein to be secreted. “Signal sequence” is used interchangeablyherein. Nearly all secreted proteins use an amino-terminal proteinextension which plays a crucial role in the targeting to andtranslocation of precursor proteins across the membrane and which isproteolytically removed by a signal peptidase during or immediatelyfollowing membrane transfer.

In preferred embodiments the signal sequence is selected fromsec-dependent signal peptides or tat-dependent signal peptides derivedfrom Bacillus.

Enhanced

The present invention is directed to the enhanced production andsecretion of a protein of interest. When discussing expression enhancedhas the following meanings. In the case a homologous protein enhancedshould be read as expression above normal levels in said host. In thecase of a heterologous protein basically any expression is, of course,enhanced.

When discussing resistance to proteolysis, enhanced means that theprotein of interest has an increased half-life when compared to analtered form of the protein of interest, e.g., untagged.

Non-Polar

As used herein, the term “non-polar” refers to the amino acid content ofthe carboxy tail of a protein. For example, when the last five aminoacids at the C-terminus are selected from nonpolar amino acid residues(A, V, L, I, P, F, W, M) the tail is considered nonpolar. If one or twoof these last five residues are uncharged polar (G, S, T, C, Y, N, Q),the tail would still be considered substantially nonpolar.

In contrast, if one or two of the last two amino acids at the C-terminusis/are charged polar: D or E (negatively charged) or K, R or H(positively charged), the tail would be considered polar, charged and,according to the present invention, this makes the protein resistantagainst proteolytic degradation by a subclass of proteases thatrecognize nonpolar C-terminal tails of secreted proteins.

Tag Sequence

As used herein, a “tag sequence” or “tag” refers to a short peptidesequence on the carboxy terminus of an expressed protein that effectsthe proteolysis of the expressed protein. For example, the bacterial tagencoded by ssrA is cotranslationally added to truncated polypeptides,thereby targeting these molecules for proteolytic degradation. It is tobe understood that in the present invention the addition of a tag servesto signal a decrease in proteolytic degradation, i.e., enhancedresistance to proteases, of proteins with substantially non-polarcarboxy termini.

Preferably the tag is at least one charged amino acid residue.Preferably, the tag comprises two charged amino acid residues. Thecharged amino acid residue(s) may be positively charged. Alternatively,the charged amino acid residue(s) may be negatively charged.

The tag should be as short as possible, since the tag itself mayinfluence the activity, specificity etc. of the protein product. Ingeneral, one can expect that a short tag of two or three amino acids hasno or just a minor effect on folding of the protein (and thereby) theactivity, specificity, etc. But there is probably no general rule forthis, it depends on the nature of the protein/enzyme.

In another preferred embodiment, the tag may be a modified Bacillus SsrAtag. In an especially preferred embodiment the modified tag has thesequence AGKTNSFNQNVALDD (SEQ ID NO:2) or AGKTNSFNQNVALKK (SEQ ID NO:4).

Isolated or Purified

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

Native Protein or Polypeptide

As used herein, the terms “Native protein” or “Native polypeptide” areused interchangeably herein and refer to a protein or polypeptide whichhas not been modified or altered at the last two amino acid residueslocated at the carboxy-terminus. In other words, the last two amino acidresidues at the carboxy-terminus of the expressed protein or polypeptideare the same as those found in the naturally occurring protein orpolypeptide. Other residues within the protein or polypeptide may bealtered, modified or mutated.

Heterologous Protein

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in a host cell. Examples ofheterologous proteins include enzymes such as hydrolases includingproteases, cellulases, amylases, other carbohydrases, and lipases;isomerases such as racemases, epimerases, tautomerases, or mutases;transferases, kinases and phophatases, hormones, growth factors,cytokines, antibodies and the like.

Homologous Protein

The term “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in a host cell. The invention includes host cellsproducing the homologous protein via recombinant DNA technology. Thepresent invention encompasses a host cell having a deletion orinterruption of the nucleic acid encoding the naturally occurringhomologous protein, such as a protease, and having nucleic acid encodingthe homologous protein re-introduced in a recombinant form. In anotherembodiment, the host cell produces the homologous protein.

Nucleic Acid Molecule.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given protein maybe produced. The present invention contemplates every possible variantnucleotide sequence, encoding tag sequences, including but not limitedto the individual amino acid residues of D, E, K and N, all of which arepossible given the degeneracy of the genetic code.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native cell.

Vector

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

Expression Cassette

Accordingly, an “expression cassette” or “expression vector” is anucleic acid construct generated recombinantly or synthetically, with aseries of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

Plasmid

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in many bacteria andsome eukaryotes.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin mammalian cells and where expression of the selectable marker confersto cells containing the expressed gene the ability to grow in thepresence of a corresponding selective agent.

Promoter

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences.

“Chimeric gene” or “heterologous nucleic acid construct”, as definedherein refers to a non-native gene (i.e., one that has been introducedinto a host) that may be composed of parts of different genes, includingregulatory elements. A chimeric gene construct for transformation of ahost cell is typically composed of a transcriptional regulatory region(promoter) operably linked to a heterologous protein coding sequence,or, in a selectable marker chimeric gene, to a selectable marker geneencoding a protein conferring antibiotic resistance to transformedcells. A typical chimeric gene of the present invention, fortransformation into a host cell, includes a transcriptional regulatoryregion that is constitutive or inducible, a signal peptide codingsequence, a protein coding sequence, and a terminator sequence. Achimeric gene construct may also include a second DNA sequence encodinga signal peptide if secretion of the target protein is desired.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader, i.e., a signal peptide, is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

The gene may encode therapeutically significant proteins or peptides,such as growth factors, cytokines, ligands, receptors and inhibitors, aswell as vaccines and antibodies. The gene may encode commerciallyimportant industrial proteins or peptides, such as enzymes, e.g.,proteases, carbohydrases such as amylases and glucoamylases, cellulases,oxidases and lipases. The gene of interest may be a naturally occurringgene, a mutated gene or a synthetic gene.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe; whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al., 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5× Denhardt's solution, 0.5%SDS and 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through two or more generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

Proteolysis in bacteria serves to rid the cell of abnormal and misfoldedproteins. A unique mechanism for the destruction of abnormal proteinsresulting from abortive termination of translation is provided by theSsrA-mediated tagging and degradation system (for a recent review, seeKarzai et al. 2000. The SsrA-SmpB system for protein tagging, directeddegradation and ribosome rescue. Nat. Struct. Biol. 7:449-455). SsrA,also called 10sa RNA or tmRNA is a highly conserved RNA molecule ineubacteria. It is a unique molecule that can act as both a tRNA and anmRNA in a process referred to as trans-translation (Atkins et al. 1996.A case for trans translation. Nature 379:769-771; Jentsch 1996. Whenproteins receive deadly messages at birth. Science 271:955-956; Keileret al. 1996. Role of a peptide tagging system in degradation of proteinssynthesized from damaged messenger RNA. Science 271:990-993). Thismechanism provides the cell a way to release ribosomes that are stalledon untranslatable mRNAs, e.g. mRNAs lacking in-frame stop codons. In themodel for SsrA action, SsrA charged with alanine enters the A site of astalled ribosome, mimicking a tRNA. The alanine is added to theuncompleted polypeptide chain; and then, serving as an mRNA, SsrAprovides a short reading frame followed by a stop codon as a template toadd a short peptide to the nascent polypeptide before translationterminates and a tagged protein is released. The peptide tag (encoded bySsrA) functions as a proteolytic degradation signal, and in Escherichiacoli four proteases have been identified that degrade proteins tagged bySsrA. ClpXP, ClpAP, and FtsH (HflB) degrade SsrA tagged proteins in thecytoplasm (Gottesman et al. 1998. The ClpXP and ClpAP proteases degradeproteins with carboxy-terminal peptide tails added by the SsrA-taggingsystem. Genes Dev. 12:1338-1347; Herman et al. 1998. Degradation ofcarboxy-terminal-tagged cytoplasmic proteins by the Escherichia coliprotease HflB (FtsH). Genes Dev. 12:1348-1355), while SsrA taggedproteins with signal peptides that are exported to the periplasm of E.coli are degraded by Tsp (Prc) protease (Keiler et al. 1996).

Not only ribosome stalling on messages without in-frame stop codonsleads to activation of the SsrA tagging system. It also occurs whenribosomes stall at clusters of rare codons in an mRNA when the cognatetRNA is scarce (Roche et al. 1999. SsrA-mediated peptide tagging causedby rare codons and tRNA scarcity. EMBO J. 18:4579-4589), and there maybe more conditions that result in SsrA tagging. The whole story leadingto the elucidation of SsrA function started with the observation by Tuet al. (1995. C-terminal extension of truncated recombinant proteins inEscherichia coli with a 10Sa RNA decapeptide. J. Biol. Chem.270:9322-9326) that a fraction of mouse interleukin-6 expressed in E.coli is truncated and contained the SsrA tag. It is not clear why inthis case part of the mIL-6 molecules were tagged by SsrA. Perhaps mIL-6mRNA is relatively unstable in E. coli, leading to transcripts that aretrimmed at the 3′ end by nucleases, thereby losing its stop codon.Alternatively, mIL-6 overexpression itself may lead to jamming at theribosomes, thereby activating the SsrA tagging system. Whatever thereason is, contamination of recombinant proteins with molecules that aretruncated and tagged by the SsrA system (and escape from degradation)restricts the usefulness of these molecules e.g. as pharmaceuticalproteins. Therefore, peptide tagging according to the present invention,i.e., the utilization of a charged tag, in B. subtilis, an industriallyimportant species used for the commercial production of various proteinsprovides a substantial benefit not found in the prior art.

B. subtilis SsrA has been isolated and sequenced several years ago(Ushida et al. 1994 tRNA-like structures in 10Sa RNAs of Mycoplasmacapricolum and Bacillus subtilis. Nucleic Acids Res. 22:3392-3396) andthe sequence of the proteolysis tag encoded by B. subtilis SsrA((A)GKTNSFNQNVALAA SEQ ID NO:1) has been predicted (Williams 2000. ThetmRNA website. Nucleic Acids Res. 27:165-166). Recently, Wiegert andSchumann (2001. SsrA-mediated tagging in Bacillus subtilis. J.Bacteriol. 183:3885-3889) showed that the CIpXP protease is responsiblefor the degradation of intracellular SsrA-tagged proteins in B.subtilis. The instant invention provides for an enhanced proteinstability via enhance protease resistance. In particular, theextracellular protease CtpA, and perhaps one or more of the majorextracellular proteases of B. subtilis, play a role in the degradationof an extracellular, heterologous protein that was tagged by the SsrAsystem. It is a benefit that tagged proteins according to the presentinvention are more resistant to proteolysis.

Possible Signal Sequences that May be Used

It is contemplated that any signal sequence that directs the nascentpolypeptide into a secretory pathway may be used in the presentinvention. It is to be understood that as new signal sequences arediscovered that they will be encompassed by the invention.

Signal peptides from two secretory pathways are specificallycontemplated by the instant invention. The first pathway is thesec-dependent pathway. This pathway is well characterized and a numberof putative signal sequences have been described. It is intended thatall sec-dependent signal peptides are to be encompassed by the presentinvention. Specific examples include but are not limited to the AmyL andthe AprE sequences. The AmyL sequence refers to the signal sequence forα-amylase and AprE refers to the AprE signal peptide sequence [AprE issubtilisin (also called alkaline protease) of B. subtilis].

The second pathway is the twin arginine translocation or Tat pathway.Similarly, it is intended that all tat-dependent signal peptides are tobe encompassed by the present invention. Specific examples include butare not limited to the phoD and the lipA sequences.

Possible Proteins that May be Produced

The present invention is particularly useful in enhancing the productionand secretion of proteins that possess non-polar or substantiallynon-polar carboxy termini. Thus, it is contemplated that a protein thatcomprises a signal sequence and a non-polar or substantially non-polarcarboxy terminus would be useful in the present invention. The proteinmay be homologous or heterologous. Proteins that may produced by theinstant invention include, but are not limited to, hormones, enzymes,growth factors, cytokines, antibodies and the like.

Enzymes include, but are not limited to, hydrolases, such as protease,esterase, lipase, phenol oxidase, permease, amylase, pullulanase,cellulase, glucose isomerase, laccase and protein disulfide isomerase.

Hormones include, but are not limited to, follicle-stimulating hormone,luteinizing hormone, corticotropin-releasing factor, somatostatin,gonadotropin hormone, vasopressin, oxytocin, erythropoietin, insulin andthe like.

Growth factors are proteins that bind to receptors on the cell surface,with the primary result of activating cellular proliferation and/ordifferentiation. Growth factors include, but are not limited to,platelet-derived growth factor, epidermal growth factor, nerve growthfactor, fibroblast growth factors, insulin-like growth factors,transforming growth factors and the like.

Cytokines are a unique family of growth factors. Secreted primarily fromleukocytes, cytokines stimulate both the humoral and cellular immuneresponses, as well as the activation of phagocytic cells. Cytokinesinclude, but are not limited to, colony stimulating factors, theinterleukins (IL-1 (α and β), IL-2 through IL-13) and the interferons(α, β and γ).

Human Interleukin-3 (IL-3) is a 15 kDa protein containing 133 amino acidresidues. IL-3 is a species specific colony stimulating factor whichstimulates colony formation of megakaryocytes, neutrophils, and macrophages from bone marrow cultures.

Antibodies include, but are not limited to, immunoglobulins from anyspecies from which it is desirable to produce large quantities. It isespecially preferred that the antibodies are human antibodies.Immunoglobulins may be from any class, i.e., G, A, M, E or D.

Possible Tags That May be Used

Tags may either be added to the carboxy terminus of a protein orsubstituted for the amino acids of the protein's carboxy terminus. Ifthe protein has been tagged by the addition of amino acid residues thetag is preferably up to 20 additional residues preferably about 15, morepreferred 1-14, even more preferred 1-11, and most preferred 1-3,wherein the last one or two amino acid residues are charged. FIG. 7Ddepicts a protein with a tag added on to its carboxy terminus (SEQ IDNO:28). In this depiction the tag is 14 amino acid residues long.

In the alternative, the tag may replace between 1 and 5 amino acids inthe protein's carboxy terminus. In the substituted tag the amino acidsare charged. In a preferred embodiment the last 5 amino acids arereplaced with the tag. In another preferred embodiment the last 4 aminoacids are replaced with the tag. In yet another preferred embodiment thelast 3 amino acids are replaced with the tag. In a more preferredembodiment the last amino acid is replaced with the tag. In a mostpreferred embodiment the last 2 amino acids are replaced with the tag.FIG. 7C depicts a substitution tagged protein (SEQ ID NO:27). In thisdepiction the final two amino acid residues of the native protein havebeen replaced with two charged amino acid residues.

The charged amino acid residues may be either positively or negativelycharged. The preferred negatively charged amino acids are: D or E. Thepreferred postively charged amino acids are: K, R or H.

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and practice the presentinvention. They should not be considered as limiting the scope and/orspirit of the invention, but merely as being illustrative andrepresentative thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); pmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC(thin layer achromatography); SDS-PAGE (Sodium dodecylsulfatepolyacrylamide gel electophoresis); Ts (tosyl); Bn (benzyl); Ph(phenyl); Ms (mesyl); Et (ethyl), Me (methyl).

EXAMPLE 1 Plasmid Construction and Host Transformation

To study the degradation of SsrA-tagged proteins in B. subtilis,variants of pLATIL3 were made in which h-IL3 is expressed with differentshort peptide tags added to the COOH-terminus of h-IL3:

-   -   Variant 1: plasmid pLATIL3-BStag; expresses the hIL3 variant        hIL3-AA: hIL3 with an apolar C-terminal B. subtilis SsrA-tag        [GKTNSFNQNVALAA] (SEQ ID NO:5)    -   Variant 2: plasmid pLATIL3-DDtag; expresses the hIL3 variant        hIL3-DD: hIL3 with a negatively charged C-terminal tag        [GKTNSFNQNVALDD] (SEQ ID NO:6), this variant differs from        variant 1 (hIL3-AA) only in the last two C-terminal amino acids        (two aspartic acids (DD) instead of two alanine (AA) residues)    -   Variant 3: plasmid pLATIL3-Ectag; expresses the hIL3 variant        hIL3-EOAA: hIL3 with an apolar C-terminal E. coil SsrA tag        [AANDENYALAA] (SEQ ID NO:3)

Plasmids, bacterial strains and media. Table I lists the plasmids andbacterial strains used in this study. E. coli strains were grown in oron 2×YT medium (Bacto tryptone, 16 g/l; yeast extract, 10 g/l; and NaCl,5 g/l). B. subtilis strains were grown in TSB (Tryptone Soya Broth fromOxoid, 30 g/l), or 2×SSM (Spizizen's minimal medium; Harwood et al. 1990Molecular biological methods for Bacillus. John Wiley and Sons,Chichester, United Kingdom), or on SMA (Spizizen's minimal agar; Harwoodet al. 1990), or HI-agar (Heart Infusion agar from Difco, 40 g/l)., Whenappropriate, media were supplemented with ampicillin, 100 μg/ml;chloramphenicol, 5 μg/ml; erythromycin, 1 μg/ml; neomycin, 10 μg/ml;spectinomycin, 100 μg/ml; tetracycline, 10 μg/ml and/orisopropyl-β-D-thiogalactopyranoside (IPTG; 500 μM).

Plasmid DNA was isolated with the QIAprep spin miniprep kit (Qiagen)according to the instructions, except that B. subtilis cells wereincubated with lysozyme (5 mg/ml in buffer P1) for 10′ at 37° C. priorto addition of lysis buffer (buffer P2). Chromosomal DNA was isolated asdescribed previously (Harwood et al. 1990). Procedures for DNArestriction, ligation, agarose gel electrophoresis, and transformationof E. coli were carried out as described in Sambrook et al., (1989.Molecular Cloning: A laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Enzymes were from Lifetechnologies.

Transformation of competent cells was used to transfer DNA (plasmids,linear DNA) into B. subtilis (Harwood et al. 1990). PCR (polymerasechain reaction) was carried out with High Fidelity Platinum Taq DNAPolymerase (Life technologies) and if required PCR fragments werepurified with the Qiaquick PCR purification kit (Qiagen). DNA primerswere from Life technologies and DNA sequencing was performed byBaseClear (Leiden, The Netherlands). Plasmid pLATIL3TERM was obtained byPCR on pLATIL3 with the primers pLATIL3SXHfw (5′ GTC GAC CTC GAG ACC CCAAGC TTG GCG TAA TC 3′) (SEQ ID NO:7) and pLATIL3T3rv (5′ GTC GAC CTC GAGCGG CAG AAT CTT TTT TTG ATT CTG CCG CAA AGT CGT CTG TTG AGC CTG 3′) (SEQID NO:8). The resulting DNA fragment was purified, digested with Xhol,self-ligated, and transformed directly into B. subtilis. One of theplasmid clones, found to be correct by DNA sequencing, was designatedpLATIL3TERM. This plasmid holds the transcription terminator of the folCgene (present in primer pLATIL3T3rv) at the 3′end of the AmyL-hIL-3gene, just in front of an in-frame stop codon. Plasmid pLATILBStag wasobtained by a PCR on pLATIL3 with the primers pLATIL3T2FW (5′ CTG CAGCTC GAG GAT ATC GTC GAC CGG CAG AAT CAA AAA AAG ATT CTG CCG ACC CCA AGCTTG GCG TAA TC 3′) (SEQ ID NO:9) and pIL3BStagRV (5′ CTT CTA CTC GAG TCAGGC AGC TAA TGC TAC GTT TTG GTT AAA ACT GTT AGT TTT GCC TGC GCT CAA AGTCGT CTG TTG AGC 3′) (SEQ ID NO:10). The resulting PCR fragment waspurified, digested with Xhol, self-ligated, and transformed into B.subtilis. A few clones were checked by DNA sequencing and one correctclone was selected and named pLATIL3BStag. Plasmid pLATIL3DDtag andpLATIL3ECtag were made in the same way but instead of primerpIL3BstagRV, primer pIL3DDtagRV (5′ CTT CTA CTC GAG TCA GTC GTC TAA TGCTAC GTT TTG GTT AAA ACT GTT AGT TTT GCC TGC GCT CAA AGT CGT CTG TTG AGC3′) (SEQ ID NO:11) and primer pIL3EctagRV (5′ CTT CTA CTC GAG TCA AGCTGC TAA AGC GTA GTT TTC GTC GTT TGC TGC GCT CAA AGT CGT CTG TTG AGC 3′)(SEQ ID NO:12) were used, respectively. To construct B. subtilis ΔssrAmutants, ssrA and its flanking regions (approximately 2.2 kb) wasamplified by PCR with the primers pSsrAFW (5′ CAG CTC CGT CTG AGG AAAAAG 3′) (SEQ ID NO:13) and pSsrARV (5′ CGA AGT GGG CGA TTT CTT CCG 3′)(SEQ ID NO:14) and cloned into pCR2.1-TOPO, resulting in plasmidpTPSsrA. Plasmid pSsrASp was obtained by inserting a pDG1726-derived Spresistance marker (Guérout-Fluery et al. 1995. Antibiotic-resistancecassettes for Bacillus subtilis. Gene 167:335-336) into the unique Saclsite in the ssrA gene of pTPSsrA. Finally, B. subtilis 168 ΔssrA andWB600 ΔssrA were obtained by a double cross-over recombination eventbetween the disrupted ssrA gene of pSsrASp and the chromosomal ssrA genein B. subtilis 168 and WB600, respectively. SsrA^(DD) expressing B.subtilis strains were made as follows: a fragment consisting of a 5′ endpart of ssrA including the ssrA promoter region, was amplified with theprimers pSsrAHindlllfw (5′ TTC TAA AAG CTT AGT GCT TGA TTC GAA AAT CAGGCC TGT G 3′) (SEQ ID NO:15 and pSsrADDintRV (5′ GAG CTC GCT GCG CTT ATTAGT CGT CTA ATG CTA CGT TTT GGT TAA 3′) (SEQ ID NO:16); contains thealteration of the two alanine codons in the SsrA tag sequence intocodons for aspartic acid residues). In addition, an overlapping 3′endpart of ssrA was amplified with the primers pSSrADDintFW (5′ TTA ACC AAAACG TAG CAT TAG ACG ACT AAT AAG CGC AGC GAG CTC 3′) (SEQ ID NO:17); alsocontaining the alteration of the two alanine codons into codons for twoaspartic acid residues) and pSsrASphlRV (5′ CCT CCG TGC ATG CTT CCT CTTATT TAT TGA CAG AAA TCT G 3′) (SEQ ID NO:18). Both fragments wereassembled in a fusion PCR with primers pSsrAHindIIIFW and pSsrASphlRV,and cloned in pCR2.1-TOPO, resulting in plasmid pSsrADD. The correctsequence of the fusion product in pSsrADD was confirmed by DNAsequencing. Next, a selective marker (the Tc resistance cassette derivedfrom pDG1515; Guérout-Fluery et al. 1995. Antibiotic-resistancecassettes for Bacillus subtilis. Gene 167:335-336) that functions in B.subtilis, was cloned into the EcoRV site of pSsrADD, resulting inplasmid pSsrADDTc. Finally, B. subtilis 168 IssrA^(DD) and WB600IssrA^(DD) were obtained by a Oampbell-type integration (singlecross-over) of pSsrADDTc into one of the disrupted ssrA regions on thechromosome of B. subtilis 168 ΔssrA and WB600 ΔssrA, respectively. Thesestrains contain an active copy of the ssrA^(DD) gene on the chromosome(under control of the native ssrA promoter) and a disrupted copy ofwild-type ssrA (insertion of the Sp resistance marker), as confirmed byPCR. To construct B. subtilis WB600 ΔctpA, WB600 was transformed withchromosomal DNA of BSE-23. In BSE-23, the ctpA gene is replaced by aspectinomycin resistance cassette (Edwin Lee, Genencor InternationalPalo Alto, unpublished). WB600 ΔyvjB was obtained as follows: yvjB andits flanking regions (approximately 3.5 kb) was amplified by PCR withthe primers pYvjBFW (5′ AGA GTT TTA AAT CTC TCG GGA GAA ACA CAT GGA TGACAT T 3′) (SEQ ID NO:19) and pYvjBRV (5′ TGT ATA TGT AAA TTT CAG ATC ATCATA AAT ATC TGC TAT T 3′) (SEQ ID NO:20) and cloned in pCR2.1-TOPO,resulting in plasmid pTPYvjB. Plasmid pTPYvjBTc was obtained byreplacing an internal SmaI-AccI fragment of the yvjB gene in pTPYvjBwith a pDG1515-derived Tc resistance marker (Guérout-Fluery et al. 1995.Antibiotic-resistance cassettes for Bacillus subtilis. Gene167:335-336). Finally, B. subtilis WB600 ΔyvjB was obtained by a doublecross-over recombination event between the disrupted yvjB gene ofpTPYvjBTc and the chromosomal yvjB gene. To construct B. subtilis WB600IcIpP, the 5′ end region of the clpP gene was amplified by PCR with theprimers pCIpPEcoFW (5′ CTT ACC GAA TTC GTG AAG GAG GAG CAT TAT G 3′)(SEQ ID NO:21) containing a EcoRI site, and pClpPBamRV (5′ GCC TTT GGATCC GGC TGC AAG CAG GAA CGC 3′) (SEQ ID NO:22) containing a BainHI site.The amplified fragment was cleaved with EcoRI and BamHI, and cloned inthe corresponding sites of pMutin2 (Vagner et al. 1998. A vector forsystematic gene inactivation in Bacillus subtilis. Microbiology144:3097-3104), resulting in plasmid pMutCIpP. B. subtilis WB600 lclpPwas obtained by a Campbell-type integration (single cross-over) ofpMutClpP into the clpP region on the chromosome. Cells of this strainare depleted for CIpP by growing them in medium without IPTG (Vagner etal. 1998).

TABLE 1 Plasmids and Strains Plasmid/Strain Properties Reference pLATIL3derivative of pGB/IL-322: contains Van Leen et al. 1991. the human IL-3gene fused to the Production of human sequence encoding the signalinterleukin-3 using peptide of B. licheniformis α- industrialmicroorganisms. amylase (amyL-hIL-3); the amyL-hIL- Biotechnology 9:47-52. 3 gene fusion is under control of the amylase promoter; 4.3 kb;Nm^(R) pLATIL3TERM derivative of pLATIL3; contains the This worktranscription terminator of the B. subtilis folC gene inserted just infront of the stop codon of amyL-hIL- 3; 4.1 kb; Nm^(R) pLATIL3BStagderivative of pLATIL3; contains This work amyL-hIL-3 fused at the 3'endto the sequence encoding the B. subtilis SsrA peptide tag(AGKTNSFNQNVALAA) SEQ ID NO:1); 4.2 kb; Nm^(R) pLATIL3DDtag derivativeof pLATIL3; contains This work amyL-hIL-3 fused at the 3'end to thesequence encoding a variant SsrA- DD-tag (AGKTNSFNQNVALDD) SEQ ID NO:2);4.2 kb Nm^(R) pLATIL3ECtag derivative of pLATIL3; contains This workamyL-hIL-3 fused at the 3'end to the sequence encoding the E. coli SsrApeptide tag (AANDENYALAA) SEQ ID NO:3); 4.2 kb Nm^(R) pCR2.1-TOPO TAcloning vector for PCR products; Invitrogen 3.9 kb; Ap^(R); Km^(R)pTPSsrA pCR2.1-TOPO derivative; carrying This work the ssrA gene +flanking regions; 6.1 kb; Ap^(R); Km^(R) pSsrASp derivative of pTPSsrAfor the This work disruption of ssrA; 7.0 kb; Ap^(R); Km^(R); Sp^(R)pSsrADD pCR2.1-TOPO derivative; carrying a This work ssrA^(DD) genevariant: the last two codons of the tag sequence in ssrA (gct gcc)encoding two alanines are changed into gac gac, encoding two asparticacid residues; 4.6 kb; Ap^(R); Km^(R) pSsrADDTc derivative of pSsrADD;carrying This work ssrA^(DD) and a Tc resistance cassette; forintegration of ssrA^(DD) on the B. subtilis chromosome; 6.8 kb; Ap^(R);Km^(R); Tc^(R) pTPYvjB pCR2.1-TOPO derivative; carrying This work theyvjB gene + flanking regions; 7.4 kb; Ap^(R); Km^(R) pTPYvjBTcderivative of pTPYvjB for the This work disruption of yvjB; 8.9 kb;Ap^(R); Km^(R); Tc^(R) pMutin2 pBR322-based integration vector forVagner et al. 1998. A B. subtilis; containing a multiple vector forsystematic gene cloning site downstream of the inactivation in BacillusPspac promoter, and a promoter-less subtilis. Microbiology 1998 lacZgene preceded by the 144: 3097-3104. RBS of the spoVG gene; 8.6 kb;Ap^(R); Em^(R) pMutClpP pMutin2 derivative; carrying the 5′ This workpart of the B. subtilis clpP gene; 8.9 kb; Ap^(R); Em^(R) Strains E.coli TOP10 F mcrA Δ(mrr-hsdRMS-mcrBC) Invitrogen Φ80lacZΔM15 ΔlacX74recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (Str^(R)) endA1 nupGXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 Stratagene supE44 relA1 lac[F¢proAB lacl^(q)ZDM15 Tn10 (Tet^(r))] B. subtilis 168 trpC2 Kunst etal. 1997. The complete genome sequence of the Gram- positive bacteriumBacillus subtilis. Nature 390: 249-256. 168 ΔssrA trpC2, ssrA; Sp^(R)This work 168 IssrA^(DD) trpC2, IssrA^(DD); Tc^(R); integration of Thiswork pSsrADDTc in ssrA::spec in 168 ΔssrA WB600 trpC, nprE, aprE, epr,bpf, mpr, Wu et al. 1991. nprB Engineering a Bacillus subtilisexpression- secretion system with a strain deficient in sixextracellular proteases. J. Bacteriol. 173: 4952-4958. BSE-23 ctpA;Sp^(R) E. Lee, unpublished WB600 ΔctpA trpC, nprE, aprE, epr, bpf, mpr,This work nprB, ctpA; Sp^(R) WB600 ΔyvjB trpC, nprE, aprE, epr, bpf,mpr, This work nprB yvjB; Tc^(R) WB600 IclpP trpC, nprE, aprE, epr, bpf,mpr, This work nprB, Pspac-clpP; clpP-lacZ; Em^(R) WB600 ΔssrA trpC,nprE, aprE, epr, bpf, mpr, This work nprB, ssrA; Sp^(R) WB600 IssrA^(DD)trpC, nprE, aprE, epr, bpf, mpr, This work nprB, IssrA^(DD); Tc^(R)

EXAMPLE 2 IL-3 Expression

When fused to the signal peptide of B. licheniformis α-amylase, humaninterleukin-3 can be secreted by B. subtilis (Van Leen et al. (1991),Biotechnology, 9: 47-52). Plasmid pLATIL3 contains the h-IL3 gene fusedto the coding region of the B. licheniformis α-amylase (AmyL) signalpeptide; In this plasmid expression of the hybrid AmyL-hIL3 gene iscontrolled by the B. licheniformis α-amylase promoter. During secretion,the AmyL signal peptide is removed from the AmyL-hIL3 precursor bysignal peptidases, and mature hIL-3 is released into the medium.

Expression of the human IL-3 gene lacking an in-frame stop codon inwild-type B. subtilis and in an ssrA mutant. Mutant 168 ΔssrA wascreated, in which the ssrA gene is disrupted by insertion of aspectinomycin resistance cassette. The mutation was checked by PCR, andthe absence of SsrA RNA in the mutant was confirmed by Northern blotanalysis (FIG. 1A). Growth of 168 ΔssrA was somewhat reduced compared tothe wild-type strain (FIG. 1B), as reported recently by Muto et al.(2000. Requirement of transfer-messenger RNA for the growth of Bacillussubtilis under stresses. Genes Cells 5:627-635). They also observed thatgrowth rates of cells without SsrA decreased with elevating temperatures(>45° C.). In addition, our results show that growth is more affected atlow temperatures (<25° C.) then at temperatures between 30-45° C. (FIG.2C), indicating a mild cold-sensitivity of growth in mutant 168 ΔssrA.

Plasmid pLATIL3, a derivative of pGB/IL-322, contains an expressioncassette for the production of human interleukin-3 (hIL-3) by Bacilli(Van Leen et al. 1991). In this construct, the B. licheniformisα-amylase (AmyL) signal peptide is used to direct secretion of maturehIL-3. As a model for SsrA-mediated peptide tagging in B. subtilis , avariant of plasmid pLATIL3 was created in which a transcriptionterminator is inserted into the AmyL-hIL3 gene, just in front of itsstop codon. Transformation of this plasmid (pLATIL3TERM) into B.subtilis will result in AmyL-hIL3 transcripts lacking in-frame stopcodons. According to the tmRNA model for SsrA mediated tagging ofproteins (Keiler et al. 1996), translation of these transcript willresult in ribosome stalling, and subsequently recruitment of SsrA,peptide tagging, and finally degradation of the tagged hIL-3 moleculesby specific proteases. To test this model in Bacillus, the extracellularproteins produced in cultures of B. subtilis 168 (pLATIL3TERM), 168ΔssrA (pLATIL3TERM), and the control strain 168 (PLATIL3), were analyzedby Western blotting (FIG. 2). Human IL-3 accumulated in the medium ofstrain 168 ΔssrA (pLATIL3TERM), but could not be detected in the mediumof B. subtilis 168 (pLATIL3TERM) containing functional SsrA. These dataindicate that B. subtilis SsrA has a role in a process in which proteinstranslated from mRNAs lacking an in-frame stop codon are degraded. Incontrast, in cells without SsrA the hIL-3 molecules are released fromstalled ribosomes by an SsrA-independent mechanism (see below). Thesemolecules do not receive a peptide-tag and, therefore are not rapidlydegraded by B. subtilis .

RNA isolation and Northern blotting. RNA was isolated with the TRIzolmethod according to the protocol provided by the manufacturer (Lifetechnologies), but with one modification: cells were incubated for 10min at 37° with lysozyme (2 mg/mI) prior to lysis in TRizol solution.Northern blotting was performed after electrophoresis of RNA throughgels containing formaldehyde (Sambrook et al. 1989). To this purpose,Hybond-N+nylon membrane from Amersham Pharmacia Biotech was used. TheSsrA-specific probe was amplified by PCR with the primers SsrAFRWDP (5′ACG TTA CGG ATT CGA CAG GGA TGG 3′) (SEQ ID NO:23) and SsrAREVP (5′ GAGTCG AAC CCA CGT CCA GAA A 3′) (SEQ ID NO:24). Labeling of the probe,hybridization and detection was performed with the ECL direct nucleicacid labeling and detection system from Amersham Pharmacia Biotechaccording to the manufacturers instructions.

EXAMPLE 3 Enhanced Protease Resistance

A pulse-chase assay was performed with the strains B. subtilis 168(pLATIL3-BStag) and B. subtilis 168 (pLATIL3-DDtag) (FIG. 2). NB: thehIL3 variants expressed by these two strains (hIL3-M and hIL3-DD) differonly in the last two COOH-terminal amino acids: two alanine residues inhIL3-AA and two aspartic acid residues for hIL3-DD. In the pulse-chaseexperiment, the initial level (pulse 1 min; chase 0′) of extracellularhIL3-DD proved to be roughly 5 times higher than that of hIL3-AA(compare lane 1 with lane 7). In addition, hIL3-AA proteins weredegraded with half lives of <2 min, while the h-IL3-DD molecules hadhalf-lives of approximately 5 min.

Protein labeling, SDS-PAGE, and fluorography. Pulse-chase labeling of B.subtilis and SDS-PAGE was essentially as described previously (Van Dijlet al. 1991. Non-functional expression of Escherichia coli signalpeptidase I in Bacillus subtilis. J. Gen. Microbiol. 137:2073-2083).However, samples collected after chase times of 0, 5, 10, 30, and 60 minwere centrifuged for 10 seconds, and only the extracellular proteins (inthe culture supernatant) were precipitated with trichloroacetic acid(TCA) and eventually subjected to SDS-PAGE. Fluorography was performedwith Amplify fluorographic reagent (Amersham-Pharmacia Biotech). Proteinbands were quantified using the Storm PhosphorImager system (MolecularDynamics).

Western blot analysis. To obtain anti-BsSsrAtag antibodies (antibodiesthat recognize proteins with a C-terminal B. subtilis SsrA-tag),synthetic peptide AGKTNSFNQNVALAA (SEQ ID NO:1) (coupled via anamino-terminal cysteine residue to KLH carrier) was injected intorabbits (Eurogentec). Serum of the final bleed of one of the rabbits wasselected for affinity purification, and this purified serum was used inthe Western blot procedures. Antibodies against human IL-3 were mousemonoclonals (Van Leen et al. 1991. Production of human interleukin-3using industrial microorganisms. Biotechnology 9:47-52). Immunoblottingand detection was performed with alkaline phosphatase-labeled conjugateand the BM Chromogenic Western Blotting kit (Roche Diagnostics)according to the instructions of the manufacturer.

Stability of hIL-3 Variants with Different C-terminal Tags Produced byB. subtilis.

To further investigate whether the B. subtilis SsrA tag functions as adegradation signal for secreted proteins, three variants of plasmidpLATIL3 were created. Plasmid pLATIL3BStag contains a gene variantencoding hIL-3 fused at the C-terminus to the B. subtilis SsrA peptidetag (AGKTNSFNQNVALAA SEQ ID NO:1), plasmid pLATIL3ECtag contains a genevariant encoding h-IL3 fused at the C-terminus to the E. coil SsrA tag(AANDENYALAA SEQ ID NO:3). The third plasmid pLATIL3DDtag contains agene encoding h-IL3 fused at the C-terminus to the sequence encoding aDD-tag (AGKTNSFNQNVALDD SEQ ID NO:2). This tag is equal to the B.subtilis SsrA-tag (AA-tag), but instead of two alanines at the extremeC-terminus it contains two aspartic acid residues. The DD-tag wassuspected to be relatively resistant to proteolytic degradation, asobserved for E. coil (Abo et al. 2000. SsrA-mediated tagging andproteolysis of Lacl and its role in the regulation of lac operon. EMBOJ. 19:3762-3769; Roche et al. 1999). The extracellular proteins producedby cells of B. subtilis 168 containing pLATIL3, pLATIL3BStag,pLATIL3DDtag, or pLATIL3ECtag, were analyzed by Western blotting (FIG.3A). The amount of the hIL-3-DDtag present in the medium was found to beroughly 5 times higher then that of wild-type hIL-3, hIL-3-AAtag orhIL-3-ECtag. Human interleukin-3 molecules produced by wild-type B.subtilis are relatively unstable due to proteolytic degradation, and theresults represented in FIG. 3A suggest that addition of a C-terminalSsrA-tag does not lead to increased degradation of hIL-3 molecules. Itis important to note, however, that in E. coil proteins taggedcotranslationally by the SsrA system are degraded more rapidly thanproteins with essentially the same sequence in which the SsrA tag is DNAencoded (Gottesman et al. 1998. The CIpXP and CIpAP proteases degradeproteins with carboxy-terminal peptide tails added by the SsrA-taggingsystem. Genes Dev. 12:1338-1347). The results obtained with pLATIL3TERM(FIG. 2) indicate that this is also true for B. subtilis. Strikingly,addition of the DD-tag (with two charged, polar residues at the extremeC-terminus) leads to a higher level of extracellular hIL-3, indicatingthat DD-tagged hIL-3 is less susceptible to proteolytic degradation. Toexplore this further, a pulse-chase assay was performed with the B.subtilis strain 168 (pLATIL3BStag) and 168 (pLATIL3DDtag) (FIGS. 3B and3C). The initial level (chase time =0 min) of hIL-3-DDtag in the mediumis approximately 4 times higher then that of hIL-3-AAtag. In addition,the hIL-3-AAtag variant was degraded with a half-life of <2 min, whereasthe half-life of hIL-3-DDtag was somewhat increased (approximately 5min). The latter observation supports that DD-tagged hIL-3 is lesssusceptible to extracellular proteases compared to hIL-3 with an AA-tag.However, the observation that the initial level of hIL3-DDtag in themedium is considerably higher then that of hIL3-AAtag indicates thathIL3-AAtag is also subject to proteolytic degradation before themolecules reach the medium, e.g. during passage of the cell wall of B.subtilis.

EXAMPLE 4 Prolonged Half-life

Detection of cotranslationally SsrA-tagged hIL-3 secreted by WB600 andby cells expressing an SsrA^(DD) variant. To detect SsrA-tagged h-IL3molecules secreted by B. subtilis and to identify proteases that have arole in the degradation of SsrA-tagged hIL-3, two different approacheswere used. First, pLATIL3TERM was expressed in WB600, a B. subtilisstrain lacking six extracellular proteases (Wu et al. 1991. Engineeringa Bacillus subtilis expression-secretion system with a strain deficientin six extracellular proteases. J. Bacteriol. 173:4952-4958), which maybe responsible for the degradation of extracellular, SsrA-tagged hIL-3.In the medium of a culture of WB600 (pLATIL3TERM), a band was detectedreacting with antibodies against hIL-3 as well as with antibodies raisedagainst the predicted B. subtilis SsrA-tag (FIG. 4, lane 3 and 8). Thisband is absent in the medium of B. subtilis 168 (pLATIL3TERM) (lanes 2and 7) and WB600 ΔssrA (pLATIL3TERM) (lanes 4 and 9). Thus, hIL-3molecules translated from mRNAs that lack termination codons are taggedby B. subtilis SsrA. The fact that these tagged molecules react withantibodies raised against the predicted B. subtilis SsrA peptide tag(AGKTNSFNQNVALAA SEQ ID NO:1) indicates that this prediction, which wasbased on comparative sequence analysis of SsrA sequences of severalbacteria (Williams 2000), was correct. In addition, it can be concludedthat at least one of the major extracellular proteases of B. subtilis(those that are absent in WB600) plays a role in the degradation ofextracellular, SsrA-tagged h-IL3. When SsrA is absent, stalled ribosomesare released by an SsrA-independent mechanism, referred to as ‘run-offtranslation’ (Williams et al. 1999. Resuming translation on tmRNA: aunique mode of determining a reading frame. EMBO J. 18:5423-5433). Theupper band in lane 4 probably represents the run-off translation productof full-length hIL-3 mRNA from pLATIL3TERM, while the bands with lowermolecular weight are most likely degradations products thereof. It seemsthat some run-off translation product is also formed when SsrA ispresent (lane 3), but it cannot be excluded that this band is just anN-terminal degradation product of SsrA-tagged hIL-3.

As a second approach to detect SsrA-tagged proteins, we constructed B.subtilis strains that express an SsrA variant (SsrA^(DD)), in which thefinal two codons of the peptide reading frame are changed to encodeaspartic acid residues instead of alanines. As mentioned above, it wasshown in E. coil that an SsrA^(DD) variant mediates the addition of apeptide tag that does not lead to rapid degradation (Abo et al. 2000;Karzai et al. 1999. SmpB, a unique RNA-binding protein essential for thepeptide-tagging activity of SsrA (tmRNA). EMBO J. 18:3793-3799).Evaluation of the antibodies that were raised against the predicted B.subtilis SsrA tag (AGKTNSFNQNVALAA SEQ ID NO:1) showed that theyrecognize the hIL-3 fused at the C-terminus to either the wild-type tag(AA-tag) or the protease resistant DD-tag (AGKTNSFNQNVALDD SEQ ID NO:2)(data not shown). Human IL-3 molecules tagged by SsrA^(DD) andsubsequently secreted (FIG. 4, lanes 5 and 10) are indeed relativelymore stable then hIL-3 molecules tagged by wild-type SsrA (lanes 3 and8), even in the six-fold protease negative strain WB600. The level offull-length SsrA^(DD)-tagged hIL-3 in the medium is somewhat higher thenthat of (wild-type) SsrA-tagged h-IL3 (FIG. 3, compare lane 10 with lane8) and relatively few degradation products of SsrA^(DD)-tagged hIL-3were detected with anti-hIL-3 antibody (compare lane 5 with lane 3).This observation suggests that besides the major extracellular proteasesthat are deleted in WB600, one (or more) additional protease is involvedin the degradation of SsrA-tagged hIL-3. Therefore, we studied the roleof three other proteases with respect to degradation of SsrA-taggedhIL-3.

CtpA has an additional role in the degradation of SsrA-tagged hIL-3secreted by B. subtilis . Three derivatives of WB600 were constructed.One WB600 ΔctpA, carried a deletion in the ctpA gene, a homologue of theE. coli gene encoding Tsp (tail specific protease). The other two,WB600. ΔyvjB and WB600 IcIpP, carried a deletion of the yvjB gene (alsoa homologue of E. coli tsp) or the clpP gene placed under control of theIPTG-dependent Pspac promoter of pMutin2, respectively. These threestrains, together with WB600 and WB600 ΔssrA, were transformed withplasmid pLATIL3TERM, grown in TSB medium with neomycin; and culturesupernatants of cells entering the stationary phase were analyzed byWestern blotting with anti-hIL-3 antibodies and anti-Bs-SsrAtagantibodies (FIG. 5). SsrA-tagged h-IL3 could not be detected in themedium of cells lacking SsrA (FIG. 5, lanes 5 and 10), but was presentwhen cells contained functional SsrA (all other lanes). As observedpreviously (FIG. 3), it seems that some full-length, run-off translationproduct is not only formed when cells lack SsrA (FIG. 5, lane 5), butalso when SsrA is present (lanes 1-4). However, as mentioned before, itcannot be excluded that the protein bands in lanes 1-4, which have thesame mobility as full-length, run-off product (upper band in lane 5),represent a degradation product of SsrA-tagged hIL-3. Inactivation ofyvjB or clpP in WB600 did not alter the amount of SsrA-tagged hIL-3 inthe medium (compare lanes 1 and 6 with lanes 3 and 8 and lanes 4 and 9).The absence of functional CtpA in WB600, however, leads to a higheramount of SsrA-tagged hIL-3 in the medium (lanes 2 and 7), and also to alower amount of the two smallest degradation products of hIL-3 (lane 2).Thus, the protease CtpA also plays a role in the degradation ofSsrA-tagged h-IL3 secreted by B. subtilis.

EXAMPLE 5 SsrA Tagging of Native B. subtilis Proteins

B. subtilis 168 IssrA^(DD) expressing the variant SsrA^(DD) RNA,containing the protease-resistant DD-tag sequence, was analyzed byWestern blotting using the anti-Bs-SsrAtag antibodies to detect nativeproteins of B. subtilis that are tagged through the SsrA system. Ascontrols, cells of B. subtilis 168 (expressing wild-type SsrA) and 168ΔssrA were used. Samples were taken of cells that were in theexponential growth phase or in the stationary phase, and theintracellular proteins and the extracellular proteins were analyzedseparately. A large number of intracellular proteins were detected byanti-Bs-SsrAtag antibody when cells expressed SsrA^(DD) (FIG. 6, lanes 2and 8), while almost all of these bands were absent in cells expressingeither wild-type SsrA (lanes 1 and 7) or no SsrA (lanes 3 and 9). Asobserved in E. coli (Abo et al. 2000), these results indicate that manyendogenous cellular proteins were tagged by the SsrA system, resultingin chimeric proteins. The proteins with the wild-type SsrA tag (MA-tag)are subsequently degraded by proteases, while proteins with the DD-tagescape from proteolysis. While in the exponential growth phase themajority of the reacting bands were of relatively low molecular weight(lane 2), in the stationary phase a shift was observed towards proteinswith a higher molecular weight (lane 8). In the exponential growthphase, no SsrA^(DD)-tagged proteins could be detected in the medium(lane 5), while in the stationary phase only a vague smear was observed(lane 11). This is most likely due to cell lysis, by which someintracellular SsrA-tagged proteins end up in the medium. It appears thatSsrA-mediated trans-translation occurs quite frequently in normallygrowing bacilli, and most natural substrates of SsrA seem be tointracellular proteins.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A nucleic acid molecule comprising a first nucleotide sequenceencoding a signal sequence, said signal sequence being for thesec-dependent secretory pathway or for the Twin Arginine Translocationpathway, said first nucleotide sequence being operatively linked to asecond nucleotide sequence encoding a heterologous polypeptide whereinthe last two codons of the polypeptide have been replaced with codonsfor two charged amino acid residues, wherein said charged amino acidresidues are lysine, arginine, or glutamic acid.
 2. The nucleic acidmolecule of claim 1 wherein the charged amino acid residue is positivelycharged.
 3. The nucleic acid molecule of claim 2 wherein the chargedamino acid residue is lysine (K).
 4. The nucleic acid molecule of claim2 wherein the charged amino acid residue is arginine (R).
 5. The nucleicacid molecule of claim 1 wherein the charged amino acid residue isnegatively charged.
 6. The nucleic acid molecule of claim 5 wherein thecharged amino acid residue is glutamic acid (E).
 7. A nucleic acidmolecule comprising a first nucleotide sequence encoding a signalsequence, said signal sequence being for the sec-dependent secretorypathway or for the Twin Arginine Translocation pathway, said firstnucleotide sequence being operatively linked to a second nucleotidesequence encoding a heterologous polypeptide and a third nucleotidesequence encoding a tag sequence, wherein said tag sequence comprisestwo amino acids selected from lysine, arginine, aspartic acid,asparagine and glutamine.